小型SPR分析系统设计及其在环境污染检测中的应用研究
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摘要
环境污染物所分析的化学物质具有种类繁多,形态各异,且常常相互作用,发生迁移变化,含量多在痕量水平等特点,推动了基于新技术新方法的小型自动化环境污染物分析仪器的开发研究,以满足环境科学对高灵敏度、高选择性、在线、原位、高通量、现场快速分析检测技术的紧迫要求。结合免疫分析检测方法的SPR检测技术无需分离纯化、无需标记,能在线实时监测分子之间的相互作用、可确定反应物种类和浓度定量测量,具有检测灵敏度高、抗电磁干扰性能好等特点,因而在环境污染物的检测分析中具有很好的优势。
为了满足环境检测领域的上述需求,本文以TI公司的SpreetaTM传感器为核心,结合流动注射分析技术,开发了小型自动化的SPR分析系统；在对其进行性能优化之后,测试了所设计的仪器的性能,并应用作为液相色谱的检测器；最后结合免疫检测技术,实现地表水、海水中藻毒素水质环境痕量污染物的检测。
主要研究的内容及成果包括下几个方面：
(1)根据介质中电磁波传播的Maxwell方程组及其边界条件和Drude金属自由电子气模型,推导出了光激发表面等离子体共振的过程。通过解析光的衰减全反射原理,推导出Kretschmann模型的SPR传感器的反射率公式。分析了角度调制型SPR传感器各部件选择对探测精度的影响。提出了“棱镜-Ag膜-Au膜”结构式的复合膜实现SPR信号增强的设计方法,通过理论仿真设计了该复合膜结构的优化参数。结果表明,在给定传感器结构下,复合膜的总厚度为50nm,其中银膜厚度为20nm,传感器的性能最为理想。
(2)基于TI公司的Spreeta传感器TSPR1A170100,结合流动注射分析技术及虚拟仪器设计方法,构建了小型SPR分析系统,系统可完成清洗、进样、再生等生物测量功能,实现多达8个生物样品的轮流检测。系统与免疫检测技术结合,可满足环境污染物现场准确、快速测定的需求。
(3)针对构建的系统,进行系统性能优化。研究所构建SPR分析系统的温度特性,根据温度特性分析设计高精度恒温系统改善检测系统的性能。基于所构建的恒温系统及小型化SPR分析系统,研究了溶液折射率随温度变化特性,首次提出一种应用于SPR测量溶液折射率的温度补偿方法,补偿后溶液折射率测量的波动范围比未校正前的波动范围小两个数量级。针对造成SPR共振峰曲线的整体有规律或无规律的偏移的低频噪声,难以应用既有的SPR共振峰分析方法克服,从而造成连续监控曲线信噪比差的问题,首次提出一种实现非线性非平稳信号处理的HHT数字滤波器设计方法,对SPR连续监控曲线进行后处理,将信号噪声降低了3倍,而信号的峰峰值仅降低1%,大大提高了其信噪比,测量检测限相应降低了8倍。
(4)基于构件的SPR检测系统,实验评估分析相应的性能指标。并利用SPR传感器对于溶液折射率变化敏感,可应用于定量分析溶液已知被测对象成分浓度的特点,提出将SPR检测方法应用作为分析化学流动分析体系的检测器。通过SPR技术与HPLC联用实验证明了该方法可行性。结果表明SPR传感器具有适用性强、样品消耗量小、小型价廉、通用性强等特点,适合作为流动分析体系的检测器。当被分析物质对紫外检测或荧光检测不敏感,或者需要通过衍生化才可进行测量的成分,SPR检测器具有很好的优势。
(5)基于所设计的小型化SPR分析系统,结合免疫检测技术,设计了应用于地表水MC-LR检测SPR生物传感器,测试了相关性能指标,并实现地表水的实样分析。并首次提出了应用于海洋赤潮麻痹性毒素之一OA的SPR免疫分析方法,该方法以点带面,对于海洋赤潮其他毒素的SPR免疫检测分析有一定的借鉴作用。
研究结果表明,所建立应用于环境痕量污染物分析的小型自动化SPR分析系统结合免疫检测技术具有特异性强,样品分析时间短等特点；所设计的传感器经再生后可重复使用上百次,且无需任何标记,从而大大节省分析测试的成本。所研究建立的新方法提供了一种实现环境污染物现场快速检测的解决方案。
There are a great of various chemical substances in the environmental pollutants. Due to characteristics of interaction and trace level content, it promotes the development of miniature bioassay instrument with automation based on the new method and technology, which can meet the demand of detection techniques with advantages of high sensitivity, high selectivity, in situ, in-vivo, and field quick analysis in environmental science. The SPR sensor associated with immunoassay can monitor biomolecular interaction on-line, including determinations of reactant type and analyte concentration without label, separation and purification. With the virtues of high detection sensitivity, fast response, universality and anti-electromagnetism interference, it offers advantages when be applied to environmental pollutants detection.
To satisfy the needs of environmental pollutants detection, associated with the flow injection analysis technique, a miniature SPR bio-analytical instrument with automation was developed, whose kernel is SpreetaTM sensor by TI. After optimization of the instrument performance, it acted as the detector of HPLC. Finally, associated with immunoassay, the SPR biosensors were fabricated to detect the trace amount algae toxins in the surface water and seawater.
The main research achievements include the followings.
(1) According to the Maxwell equations and its boundary condition that the electromagnetic wave propagates in the medium, and the Drude model of free-electron gas in the mental, the principle that surface plasma resonance was excited by the incident light was interpreted in detail. Based on the theory of attenuated total reflection of light, the reflectance equation of Kretschmann SPR sensor was derivated. The performance of the senor influenced by each component was simulated. The complex film with the structure of'prism-Ag film-Au film'in SPR sensor was designed. Its parameters were optimized via simulation. It could be concluded that the SPR sensor with 50nm complex film, which includes 20nm gold film, provides the best performance.
(2) Based on the Spreeta sensor TSPR1A170100 by TI Ltd, the flow injection analysis technique and the design method of virtual instruments, the miniature SPR bio-analytical instrument with automation was developed. It can perform the bioassay function of clean, injection and regeneration, and detect 8 different samples in sequence. Coupled with immunoassay method, it can be well satisfy the needs of accurate, rapid, and field detection for environmental pollutions.
(3) The performance of the fabricated SPR instrument was optimized. After the temperature effect of the instrument was studied, the efficiently constant temperature regulator was developed to improve the sensor's performance. Based on the efficiently constant temperature regulator, the variation of analyte's refractive index against temperature was evaluated. Therefore, the temperature compensation method for the refraction index of the liquid determined via SPR sensor was proposed firstly. The compensated result was two degree lower than the refraction index without compensation determined by SPR sensor. To overcome the defect that the SPR formant curve interfered by the low-frequency noise, which was difficult to be avoided by traditional analysis algorithm for SPR formant curve, the HHT digital filter for the non-stationary nonlinear signal was designed and firstly applied to filter the continuous monitoring signal of SPR. It can help to evidently improve the signal noise ratio, and the detection limit was 8 lower than that without filtering.
(4) Based on the SPR bio-analytical instrument, its performance is evaluated via experiment. Since the SPR sensor is sensitive to the changes of the refractive index of the solution, it can be applied to detect concentration of the defined composition in solution. It was proposed that the SPR acted as the novel detector of the flow analysis system. The feasibility was confirmed by the experiment that SPR detector was coupled to HPLC. Result showed that it fit for the flow analysis system due to its universality, low sample consumption and cost-effectiveness. The SPR detector is of advantage to detecting the composition insensitive to ultraviolet detection or fluorescence detection, or the composition needed to be derived for determination, such as glucide, lipid, high polymer and surfactant.
(5) Coupled with the immunoassay method, the miniature SPR bio-analytical instrument with MC-LR biosensor was applied to detection the MC-LR in surface water. The performance was evaluated, and real sample of surface water was determined. Equipped with the OA biosensor, it was firstly applied to detect OA, which belong to the PSP algae in the sea. The method can be universally established to detection the other algae in the sea.
The result of the research indicates that miniature SPR bio-analytical instrument with automation can be well applied to detect trace environmental pollution directly and rapidly without cumbersome sample pretreatment due to specificity of immunosensor. In addition, the reusable immunosensor with free-labeling contributes to reducing the cost of assay. All of its characteristics can complement the shortcomings of traditional assay method, such as long assay time, high cost and labeling. Most of all, it is a promising project to monitor field samples in situ for environmental pollution detection.
为了满足环境检测领域的上述需求,本文以TI公司的SpreetaTM传感器为核心,结合流动注射分析技术,开发了小型自动化的SPR分析系统；在对其进行性能优化之后,测试了所设计的仪器的性能,并应用作为液相色谱的检测器；最后结合免疫检测技术,实现地表水、海水中藻毒素水质环境痕量污染物的检测。
主要研究的内容及成果包括下几个方面：
(1)根据介质中电磁波传播的Maxwell方程组及其边界条件和Drude金属自由电子气模型,推导出了光激发表面等离子体共振的过程。通过解析光的衰减全反射原理,推导出Kretschmann模型的SPR传感器的反射率公式。分析了角度调制型SPR传感器各部件选择对探测精度的影响。提出了“棱镜-Ag膜-Au膜”结构式的复合膜实现SPR信号增强的设计方法,通过理论仿真设计了该复合膜结构的优化参数。结果表明,在给定传感器结构下,复合膜的总厚度为50nm,其中银膜厚度为20nm,传感器的性能最为理想。
(2)基于TI公司的Spreeta传感器TSPR1A170100,结合流动注射分析技术及虚拟仪器设计方法,构建了小型SPR分析系统,系统可完成清洗、进样、再生等生物测量功能,实现多达8个生物样品的轮流检测。系统与免疫检测技术结合,可满足环境污染物现场准确、快速测定的需求。
(3)针对构建的系统,进行系统性能优化。研究所构建SPR分析系统的温度特性,根据温度特性分析设计高精度恒温系统改善检测系统的性能。基于所构建的恒温系统及小型化SPR分析系统,研究了溶液折射率随温度变化特性,首次提出一种应用于SPR测量溶液折射率的温度补偿方法,补偿后溶液折射率测量的波动范围比未校正前的波动范围小两个数量级。针对造成SPR共振峰曲线的整体有规律或无规律的偏移的低频噪声,难以应用既有的SPR共振峰分析方法克服,从而造成连续监控曲线信噪比差的问题,首次提出一种实现非线性非平稳信号处理的HHT数字滤波器设计方法,对SPR连续监控曲线进行后处理,将信号噪声降低了3倍,而信号的峰峰值仅降低1%,大大提高了其信噪比,测量检测限相应降低了8倍。
(4)基于构件的SPR检测系统,实验评估分析相应的性能指标。并利用SPR传感器对于溶液折射率变化敏感,可应用于定量分析溶液已知被测对象成分浓度的特点,提出将SPR检测方法应用作为分析化学流动分析体系的检测器。通过SPR技术与HPLC联用实验证明了该方法可行性。结果表明SPR传感器具有适用性强、样品消耗量小、小型价廉、通用性强等特点,适合作为流动分析体系的检测器。当被分析物质对紫外检测或荧光检测不敏感,或者需要通过衍生化才可进行测量的成分,SPR检测器具有很好的优势。
(5)基于所设计的小型化SPR分析系统,结合免疫检测技术,设计了应用于地表水MC-LR检测SPR生物传感器,测试了相关性能指标,并实现地表水的实样分析。并首次提出了应用于海洋赤潮麻痹性毒素之一OA的SPR免疫分析方法,该方法以点带面,对于海洋赤潮其他毒素的SPR免疫检测分析有一定的借鉴作用。
研究结果表明,所建立应用于环境痕量污染物分析的小型自动化SPR分析系统结合免疫检测技术具有特异性强,样品分析时间短等特点；所设计的传感器经再生后可重复使用上百次,且无需任何标记,从而大大节省分析测试的成本。所研究建立的新方法提供了一种实现环境污染物现场快速检测的解决方案。
There are a great of various chemical substances in the environmental pollutants. Due to characteristics of interaction and trace level content, it promotes the development of miniature bioassay instrument with automation based on the new method and technology, which can meet the demand of detection techniques with advantages of high sensitivity, high selectivity, in situ, in-vivo, and field quick analysis in environmental science. The SPR sensor associated with immunoassay can monitor biomolecular interaction on-line, including determinations of reactant type and analyte concentration without label, separation and purification. With the virtues of high detection sensitivity, fast response, universality and anti-electromagnetism interference, it offers advantages when be applied to environmental pollutants detection.
To satisfy the needs of environmental pollutants detection, associated with the flow injection analysis technique, a miniature SPR bio-analytical instrument with automation was developed, whose kernel is SpreetaTM sensor by TI. After optimization of the instrument performance, it acted as the detector of HPLC. Finally, associated with immunoassay, the SPR biosensors were fabricated to detect the trace amount algae toxins in the surface water and seawater.
The main research achievements include the followings.
(1) According to the Maxwell equations and its boundary condition that the electromagnetic wave propagates in the medium, and the Drude model of free-electron gas in the mental, the principle that surface plasma resonance was excited by the incident light was interpreted in detail. Based on the theory of attenuated total reflection of light, the reflectance equation of Kretschmann SPR sensor was derivated. The performance of the senor influenced by each component was simulated. The complex film with the structure of'prism-Ag film-Au film'in SPR sensor was designed. Its parameters were optimized via simulation. It could be concluded that the SPR sensor with 50nm complex film, which includes 20nm gold film, provides the best performance.
(2) Based on the Spreeta sensor TSPR1A170100 by TI Ltd, the flow injection analysis technique and the design method of virtual instruments, the miniature SPR bio-analytical instrument with automation was developed. It can perform the bioassay function of clean, injection and regeneration, and detect 8 different samples in sequence. Coupled with immunoassay method, it can be well satisfy the needs of accurate, rapid, and field detection for environmental pollutions.
(3) The performance of the fabricated SPR instrument was optimized. After the temperature effect of the instrument was studied, the efficiently constant temperature regulator was developed to improve the sensor's performance. Based on the efficiently constant temperature regulator, the variation of analyte's refractive index against temperature was evaluated. Therefore, the temperature compensation method for the refraction index of the liquid determined via SPR sensor was proposed firstly. The compensated result was two degree lower than the refraction index without compensation determined by SPR sensor. To overcome the defect that the SPR formant curve interfered by the low-frequency noise, which was difficult to be avoided by traditional analysis algorithm for SPR formant curve, the HHT digital filter for the non-stationary nonlinear signal was designed and firstly applied to filter the continuous monitoring signal of SPR. It can help to evidently improve the signal noise ratio, and the detection limit was 8 lower than that without filtering.
(4) Based on the SPR bio-analytical instrument, its performance is evaluated via experiment. Since the SPR sensor is sensitive to the changes of the refractive index of the solution, it can be applied to detect concentration of the defined composition in solution. It was proposed that the SPR acted as the novel detector of the flow analysis system. The feasibility was confirmed by the experiment that SPR detector was coupled to HPLC. Result showed that it fit for the flow analysis system due to its universality, low sample consumption and cost-effectiveness. The SPR detector is of advantage to detecting the composition insensitive to ultraviolet detection or fluorescence detection, or the composition needed to be derived for determination, such as glucide, lipid, high polymer and surfactant.
(5) Coupled with the immunoassay method, the miniature SPR bio-analytical instrument with MC-LR biosensor was applied to detection the MC-LR in surface water. The performance was evaluated, and real sample of surface water was determined. Equipped with the OA biosensor, it was firstly applied to detect OA, which belong to the PSP algae in the sea. The method can be universally established to detection the other algae in the sea.
The result of the research indicates that miniature SPR bio-analytical instrument with automation can be well applied to detect trace environmental pollution directly and rapidly without cumbersome sample pretreatment due to specificity of immunosensor. In addition, the reusable immunosensor with free-labeling contributes to reducing the cost of assay. All of its characteristics can complement the shortcomings of traditional assay method, such as long assay time, high cost and labeling. Most of all, it is a promising project to monitor field samples in situ for environmental pollution detection.
引文
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